Culturing Endothelial Cell Lines

iSLK-RGB-BAC16 and iSLK-RGB-K9 mutant cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS), 1 μg/ml puromycin, 250 μg/ml G418, and 1.2 mg/ml hygromycin B. Primary human umbilical vein endothelial cells (pri-HUVECs), which were used between passages 3 and 6, were isolated from the interior of the umbilical vein of human umbilical cords by digestion with collagenase (Sigma, St. Louis, MO, USA), and cultured in complete EBM-2 culture media (LONZA, Allendale, NJ, USA) as previously described [56 (link)]. pri-HUVECs were used for migration and invasion assays, a human umbilical vein endothelial cell line, EA.hy926 (catalog #CRL-2922; ATCC, Manassas, VA, USA) was employed for RNA-seq analysis, plate colony formation assay and in vivo matrigel plug assay, and HEK293T cells were used for lentivirus packaging and luciferase activity assay. Both HEK293T and EA.hy926 were maintained in DMEM supplemented with 10% fetal bovine serum (FBS). All of cell lines were authenticated by short tandem repeat profiling. Effectence transfection reagent (Qiagen, Suzhou, Jiangsu, China) and Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA) were used for the transfection of endothelial cells and HEK293T cells, respectively.